The ability to perform rapid screening tests in diagnostic medicine has been considerably facilitated by the evolving art of immunoassay. Antibodies can be raised that have exquisite specificity and sensitivity for small molecules of diagnostic interest. In combination with other reagents that have a separating or labeling function, specific antibodies can be used as part of a rapid screening test for the presence of the small molecule in a clinical sample.
Small molecules that can be measured in this way include hormones, natural metabolites, and prescription drugs. Of increasing interest is the detection of substances of abuse, particularly the inappropriate voluntary use of recreational drugs. Substances of abuse include canabinoids, tranquilizers such as barbiturates, stimulants such as amphetamines, hallucinogenic alkaloids such as cocaine and lysergic acid diethylamide (LSD), and anabolic steroids.
U.S. Pat. Nos. 4,952,336 and 5.354,693 and E.P. Patent 371,253-B relate to the detecting of analytes in the amphetamine class in an assay using fluorescent tracers and antibodies raised against amphetamine derivatives. In U.S. Pat. No. 5,328,828, an assay method is recited in which the sample is combined with reagent antibodies and protein conjugates for both amphetamine and methamphetamine. Diagnostic kits for measuring amphetamine class compounds are available commercially using the CEDIA.RTM. DAU technology of Boehringer Mannheim Corp., the fluorescence polarization immunoassay (FPIA) technology of Abbott Laboratories, the EMIT.RTM.=0 II technology of Behring Diagnostics, the COBAS.RTM. INTEGRA technology of Roche Diagnostics, and the COAT-A-COUNT.RTM. radioassay technology of Diagnostic Products Corporation.
Radioimmunoassay kits for LSD are sold in the ABUSCREEN.RTM. product line by Roche and the COAT-A-COUNT.RTM. product line . International patent application PCT/US 96/19266 provides reagents and procedures for the detection and measurement of LSD in an enzyme immunoassay. A diagnostic kit for LSD based on this technology is commercially available in the CEDIA.RTM. DAU product line. More recently, other enzyme-type immunoassays for LSD and related compounds have been developed (Hu et al., Clin. Chem. (1996) 42:S219; Webb et al., J. Forensic Sciences (1996) 41:938).
Because of the possible legal implications of a positive test result, it is important that a positive immunoassay test for a substance of abuse be verified. The touchstone verification test for small molecule drugs is mass spectroscopy--particularly when combined with gas or liquid chromatography (GC/MS or LC/MS), or tandem mass spectrometry (GC/MS/MS or LC/MS/MS). These secondary tests are complex, require expensive instrumentation and highly trained personnel, and therefore are very expensive as compared to the initial screening test. Any reduction in the proportion of false positives selected for verification reduces the overall costs of drug screening.
For the problem of cross-reactivity in commercially available amphetamine assays, see D'Nicuola et al., J. Anal. Toxicol. (1992) 16:221. A major cause of false positives in the screening immunoassay is that the detecting antibody for the analyte cross-reacts with an interfering substance that is present in the samples. Accordingly, certain technologies have been developed with a view to reducing the frequency of false positives due to cross-reactivity.
Strategies for reducing false positives in assays for small molecule drugs have been principally oriented at either improving the specificity of the detecting antibody, or neutralizing the effect of the interfering substance in the sample.
U.S. Pat. No. 3,856,469 relates to the removal of .beta.-hydroxyamine compounds from samples to be tested for amphetamine, by treating with aqueous periodate under mildly basic conditions. Modifications of the periodate treatment method have been described for reducing interference by ephedrine, pseudoephedrine, and phenylpropanolamine. See Elsohly et al., J. Anal. Toxicol. (1992) 16:109; Spiehler et al., J. Anal. Toxicol. (1993) 17:125; and Paul et al., J. Anal. Toxicol. (1994) 18:331. U.S. Pat. No. 5.573,955 describes reducing tyramine interference in amphetamine immunoassays by treating with tyramine oxidase.
U.S. Pat. No. 4,843,020 relates to a method for detecting tetrahydrocannabinol (THC) in human urine by precipitating out any melanin that might be present by treating with nitroferricyanide. U.S. Pat. No. 4,477,346 relates to a method for removing interfering substances in theophylline assays, particularly caffeine. The sample to be tested is pre-treated by liquid-liquid extraction using hydrophobic, macroreticular resin slurried in a protic solvent.
U.S. Pat. No. 5,518,887 relates to competition assays using an insolubilized analog of the substance to be measured. Two different detecting antibodies are used, one of which is selected to have less cross-reactivity for the interfering substance. The disclosure proposes that the method be used to distinguish between phencyclidine (PCP) and structurally related substance, particularly 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), a methadone metabolite. In the presence of the true analyte, the binding of both antibodies to the insolubilized analog is inhibited, whereas the interfering substance is proposed to have less effect on the antibody that is more specific. Thus, positive inhibition of both antibodies would confirm the presence of the true analyte.
Strategies have also been developed for distinguishing between related proteins. Several of these strategies involve the use of several different antibodies in the analysis, relying on a difference in epitopes attributable to amino acid sequence differences between the target analyte and the potentially cross-reacting substance.
U.S. Pat. No. 4,722,889 relates to a solid-phase sandwich-type immunoassay for human chorionic gonadotropin (hCG). A scavenger antibody is included in the reaction mixture that prevents cross-reacting protein hormones from reacting with the capture or detecting antibody.
U.S. Pat. No. 5,296,354 relates to a competition-type immunoassay that is intended to distinguish Angiotensin II from any Angiotensin III that may be present in the sample. Detection of the analyte occurs via an inhibition of the reaction between an anti-Angiotensin II antibody and a labeled Angiotensin II reagent. The reaction mixture includes an antibody more specific for Angiotensin III, which is intended to prevent any Angiotensin III from inhibiting the reaction between the anti-Angiotensin II and the labeled reagent.
U.S. Pat. No. 5,064,755 is directed to a two-site confirmatory assay. The disclosure relates a method for confirming the presence in a sample of a Chlamydia antigen, which is a complex multi-epitope antigen with variant subspecies. The confirmatory assay involves comparing the results of two different tests. The first test involves developing the sample with a detecting antibody specific for Chlamydia antigen. The second test involves treating a duplicate sample with a confirmatory antibody before the detecting antibody. A predetermined decrease in signal resulting from pretreatment with the confirmatory antibody confirms the presence of the antigen in the sample. The second antibody is chosen so as to be capable of binding a second epitope on the Chlamydia and thereby prevent the binding of the first antibody.
U.S. Pat. No. 5,308,755 is directed to an assay device for concurrently detecting an analyte and confirming the test result. The exemplary device has two fluid-flow pathways, and is illustrated for use in detecting hepatitis B surface antigen (HBsAg). The first pathway contains a binding member (such as anti-HBsAg) which participates in forming an immobilized labeled complex if analyte is present in the test sample. The second pathway contains in addition a mobile confirmatory reagent (such as anti-HBsAg) which inhibits the binding of the immobilized labeled complex. The amount of labeled analyte complex immobilized in the first pathway is related to the presence of analyte in the sample, which is confirmed if the confirmatory reagent prevents the formation of the immobilized labeled complex.
A neutralization assay for confirming HBsAg in a sample is manufactured by Sorin Biomedica (Italy) and distributed in the U.S. by Incstar Corporation. The assay is described in the Instruction Manual for the REAC-801 Confirmatory Test. In the direct determination, HBsAg is captured using a first antibody on a solid phase, and developed using a labeled second antibody that reacts against a second epitope on the HBsAg. The confirmatory test involves treating the captured HBsAg with an unlabeled, neutralizing antibody before adding the labeled antibody. The neutralizing antibody is specific for HBsAg, and blocks subsequent binding of the labeled antibody to its specific epitope on HBsAg. If the signal of the neutralized sample is significantly lower than the signal of the non-neutralized sample, the presence of HBsAg in the sample is confirmed.